Analysis of labeling of plant protoplast surface by fluorophore-conjugated lectins

Walko, R. M.; Furtula, Vesna; Nothnage, E. A.

doi:10.1007/bf01276786

Cited by 21 publications

(11 citation statements)

References 28 publications

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“…The presence of β-Dgalactose was detected by FITC-RCA, but only slightly, suggesting that this sugar is not the main component of the mucilage. On the other hand, non-germinated spores fluoresced strongly when incubated with FITCConA, showing a high concentration of α-D-mannose and α-D-glucose in the mucilage, although Con-A is specific for α-D-mannose and less so for α-D-glucose (von Sengbusch et al, 1982;Walko et al, 1987). The mucilage showed a fluorescence limited to a thin layer when it was incubated with WGA, indicating a low concentration of N-acetyl-α-(1, 4)-D-glucosamine.…”

Section: Discussionmentioning

confidence: 97%

Spore Adhesion and Cell Wall Formation in Gelidium Floridanum (Rhodophyta, Gelidiales)

2006

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The attachment of spores to a substratum is essential for their germination and, therefore, to the completion of the life cycle of the red algae. In most red algae, spores are liberated without a cell wall, within a sheath of mucilage which is responsible for their primary attachment. Utilizing fluorescent-labeled lectins, we identified carbohydrate residues and their locations in the mucilage and cell walls of spores of Gelidium floridanum. Cell wall formation and mucilage composition were studied with calcofluor, toluidine blue -O (AT-O), alcian blue (AB) and periodic acid-Schiff (PAS). In the mucilage we identified α-D mannose, α-D glucose, β-D-galactose, N-acetyl-glucosamine and N-acetyl-galactosamine. The first two sugar residues were not found in the cell wall of the germ tube but they were present on the rhizoid's cell wall indicating their importance to substrate adhesion. A cell wall is produced soon after the spore's attachment, beginning with a polar deposition of cellulose and its gradual spread around the spore as indicated by calcofluor. The cell wall matrix was positive to AB and metachromatic to AT-O, indicating acidic polysaccharides, while cellulose microfibrills were positive to PAS. A polar disorganization of the cell wall triggers the process of germination. As spores are the natural form of propagation of Gelidium, the understanding of the mechanisms of spore attachment may contribute to the cultivation of this valuable seaweed.

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Section: Discussionmentioning

confidence: 97%

Spore Adhesion and Cell Wall Formation in Gelidium Floridanum (Rhodophyta, Gelidiales)

2006

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“…This demonstrates that the fluorescent signal of the FITC-Con A on sperms is due to interaction with specific lectin-sugar residues. Once FITC-Con A binds, both the position and the intensity of the staining pattern were stable, as reported before for somatic protoplast labeling (Walko et al 1987).…”

Section: Resultsmentioning

confidence: 73%

A reliable protocol for direct detection of lectin binding sites on the plasma membrane of a single living sperm cell in maize

Sun

Kranz²,

Moscatelli

et al. 2002

Sexual Plant Reproduction

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A novel protocol for direct detection and localization of lectin binding sites on the surface of single sperm cells is presented. Fluorescence-conjugated lectins and single cell manipulation techniques were employed in this protocol. Advantages of the protocol include that sperm cell membranes are well protected and artifacts avoided by minimizing movement of the medium. Damage of the sperm membrane was reduced using a dropletstaining/washing method. Any morphological change may be readily recorded.

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“…There are contradictory reports about the influence of enzymatic treatment on lectin labeling of cell membranes. Walko et al (1987) reported that several cell-wall-degrading enzymes, including cellulysin and pectolyase Y-23, generally had no effect on lectin binding. One exception was driselase, which markedly reduced Arachis hypogaea agglutinin binding to protoplasts.…”

Section: Discussionmentioning

confidence: 98%

“…Also, the binding of several lectins to somatic protoplasts derived from suspension cultured rose cells was enhanced by increasing the calcium level in the buffer. It was concluded that the effect of calcium and other divalent cations appeared to involve the plasma membrane binding sites of the somatic protoplasts rather than the lectins (Walko et al 1987).…”

Section: Discussionmentioning

confidence: 99%

“…In higher plants, lectin-receptor interaction might be involved in pollen-stigma recognition and pollen tube growth (Southworth 1975;Clarke et al 1979). Binding of several lectins to somatic plant protoplasts derived from suspension cultured rose cells or from maize root cortex has been reported (Walko et al 1987). However, there is no information on whether lectins and their receptors are involved in fertilization and embryogenesis in higher plants and whether glycoproteins can be identified on the surface of egg cell membranes.…”

Section: Introductionmentioning

confidence: 97%

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Fluorophore-conjugated lectin labeling of the cell surface of isolated male and female gametes, central cells and synergids before and after fertilization in maize

Sun

Kranz²,

Hong-yuan

et al. 2002

Sexual Plant Reproduction

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Three fluorescein isothiocyanate (FITC)-conjugated lectins, Canavalia ensiformis agglutinin (Con A), Triticum vulgaris agglutinin (WGA) and Phaseolus vulgaris erythroagglutinin (PHA-E), were used as probes to localize sugar moieties of glycoconjugates on the cell surface of isolated maize sperm, egg, central, antipodal cells, synergids, and in vitro-and in vivo-fertilized zygotes. Fluorescence signals on the surface of the cells were due to specific binding. Calcium was necessary for WGA and PHA-E binding and enhanced Con A labeling. Differences in glycoconjugate composition of the membranes of gametes and other embryo sac component cells were found. FITC-Con A strongly labeled egg and central cells, but labeled sperm only weakly. FITC-WGA binding sites were detected on egg, but not sperm cells. Con A and WGA binding sites were equally distributed around egg and central cell protoplasts. FITC-PHA-E binding sites were not found on sperm and egg cells before fertilization. Binding sites of these lectins were located on synergids, especially on their filiform apparatus. Interestingly, WGA binding to egg cells was enhanced after fertilization, whereas PHA-E binding to egg cell membranes could only be detected after fertilization. These results suggest the occurrence of fertilization-induced changes in glycoconjugate composition of the maize egg cell membrane. An increase in the number of WGA and PHA-E binding sites was also observed on newly formed cell walls of cultured two-celled embryos derived from in vitro-produced zygotes.

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Analysis of labeling of plant protoplast surface by fluorophore-conjugated lectins

Cited by 21 publications

References 28 publications

Spore Adhesion and Cell Wall Formation in Gelidium Floridanum (Rhodophyta, Gelidiales)

Spore Adhesion and Cell Wall Formation in Gelidium Floridanum (Rhodophyta, Gelidiales)

A reliable protocol for direct detection of lectin binding sites on the plasma membrane of a single living sperm cell in maize

Fluorophore-conjugated lectin labeling of the cell surface of isolated male and female gametes, central cells and synergids before and after fertilization in maize

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